Not applicable.
Not applicable.
Not applicable.
Developments in methods of manufacturing very small devices, such as microelectronic devices, have made it possible to precisely and reproducibly make devices having features with nanometer-scale dimensions. Apart from use of such methods in microelectronic device production, similar technology has been used to make devices for handling biological materials, such as cells and macromolecules.
Microengineered bio-handling devices having structural elements with minimal dimensions ranging from tens of micrometers (the dimensions of biological cells) to nanometers (the dimensions of some biological macromolecules) have been described. This range of dimensions (nanometers to tens of micrometers) is referred to herein as xe2x80x9cmicroscale.xe2x80x9d For example, U.S. Pat. No. 5,928,880, U.S. Pat. No. 5,866,345, U.S. Pat. No. 5,744,366, U.S. Pat. No. 5,486,335, and U.S. Pat. No. 5,427,946 describe microscale devices for handling cells and biological molecules.
About half of infertility cases are attributable to sperm motility dysfunction. Diagnosing such dysfunction can be difficult. Prior art methods of assessing sperm motility include manual observation and counting of sperm using a gradated device such as a Makler chamber, a microscope, and a hand-held push-button counter. In addition to being timexe2x80x94 and laborxe2x80x94intensive, such methods are susceptible to operator-to-operator differences in operation. There is a need for sperm motility-assessing devices that are simpler to operate manually, or which can be automated.
Hemocytometry is a field of medical analysis and research wherein blood cells are analyzed using variety of techniques and devices. Basic manually-operated devices such as microscope slides with Neubauer or Makler chambers were developed over a century ago. These devices are expensive, reusable, and lack flexibility, multiple features, and disposability. Disposability is especially desirable to minimize medical personnel interaction with potentially hazardous biological specimens.
Knowledge of rates of growth and proliferation of cells (e.g., mammalian cells, yeast, fungi, and cells infected with viruses) is critical for many research and clinical applications. However, there are few, if any, analytical devices which can be used for objective measurement of growth and proliferation of cells under certain conditions, such as conditions which simulate dimensions and complexity of tissues, organs, and extracellular matrices.
A significant shortcoming of previously described microscale cellxe2x80x94 and biomoleculexe2x80x94handling devices is that bulk fluid flow and other fluid dynamic phenomena through microscale channels and spaces often interferes with measurements which the devices are intended to enable. The origin of such phenomena cannot always be determined or controlled, and can be attributable to temperature, mechanical pressure, hydrodynamic pressure, and surface tension forces, for example. Microscale biohandling devices would be significantly improved if this shortcoming could be overcome.
The invention described in this disclosure overcomes this shortcoming.
The invention relates to an apparatus for analyzing cells. The apparatus comprises a base and at least two obstacles. The base defines a void for containing a liquid medium. The void has an inlet region, an outlet region, and a differentiating region interposed between the inlet and outlet regions. The obstacles are disposed within the void and define a microscale flow path and a non-microscale flow path between the inlet and outlet regions. Preferably, the cross-sectional area of the narrowest portion of the non-microscale flow path is at least 10, 100, or 1000 times the cross-sectional area of the narrowest portion of the microscale flow path. The apparatus can comprise enough obstacles to define a plurality of microscale and non-microscale flow paths, and these can vary in size. For example, one microscale flow path can be 2, 5, or 10 or more times wider than another. Alternatively, the microscale flow paths can all have about the same width. The obstacles can, for example, be elongate members (e.g., walls) that define a microscale flow path having an approximately constant width.
The distance between the base and the more distal surface of the obstacles can be approximately constant along the length of the microscale flow path defined by the elongate members. Alternatively, this distance can vary (continuously, semi-continuously, or in a stepwise fashion) along the length of the microscale flow path.
The precise construction of the obstacles is not critical. They can, for example, be connected to the base (or cover) at one or several points, or along the entire length of the obstacles. They can be connected by projections extending from one of the base (or cover) and the obstacles. In another embodiment, the obstacles are integral with the base (or cover).
The apparatus can be used with or without a cover that covers at least a portion of the void. When the apparatus has a cover, the obstacles can be connected to either or both of the cover and the base. Also, when a cover is used, at least one of the base and the cover is transparent. The cover or base can have gradations marked, imprinted, engraved, or otherwise associated therewith along a microscale flow path. The cover can have one or mole holes or ports therein for providing fluid to and withdrawing fluid from the apparatus, and those holes or ports can be adapted to fit or receive a fluid handling device (pump, tube, pipe, pippettor, etc.).
In another aspect, the invention relates to an apparatus for assessing the motility of cells in a sample. This apparatus comprises a unitary body having a surface with depressed portions. The depressed portions define
a) an inlet region for receiving the sample;
b) an outlet region for containing a fluid medium;
c) a microscale channel fluidly connecting the inlet and outlet regions, for facilitating movement of cells between the inlet and outlet regions; and
d) a non-microscale channel fluidly connecting the inlet and outlet regions for facilitating bulk fluid movement.
The surface can further define
e) a substantially flat upper surface bordering each of the microscale and non-microscale channels.
This surface can be used for receiving a substantially flat cover opposed against the upper surface. Alternatively, the surface can further define
e) a plurality of projections extending from each of the microscale and non-microscale channels.
Those projections can be used for receiving a substantially flat cover opposed against the projections. The features of this apparatus can otherwise be like the corresponding features of the apparatus described above.
The invention includes a method of assessing cell motility in a sample containing cells. The method comprises applying the sample to the inlet region of an apparatus described herein. Movement of cells from the inlet region to the outlet region by way of the microscale flow path is assessed in order to assess the cells"" motility. In this method, convective flow of the fluid medium in the void is preferably equilibrated or halted prior to applying the sample to the inlet region. This method is useful, for example, for assesing motility of human, equine, bovine, sheep, goat, canine, and feline spermatozoa.
The invention also includes a method of separating more motile cells from less motile cells in a sample. The method comprises applying the sample to the inlet region of an apparatus described herein. More motile cells are more likely than less motile cells to move from the inlet region to the outlet region. More motile cells are collected from the outlet region.
In yet another aspect, the invention includes a method of assessing proliferation of cells in a sample. This method comprises applying the sample to the inlet region of an apparatus described herein. Cells in the inlet region can proliferate and give rise to cells which are generated along the microscale flow path. The presence of cells along the microscale flow path is assessed in order to assess proliferation of the cells.
The invention further relates to a method of assessing motile cells in a sample obtained from the blood of an animal. This method comprises applying the sample to the inlet region of an apparatus described herein. Motile cells in the sample move along the microscale flow path. The presence of cells is assessed along the microscale flow path as an indication of the presence of motile cells in the sample.